NEMO, a non-catalytically active component of the IkappaB alpha kinase (IKK) complex, is required for IKK enzymatic activity and localization. If NEMO is defective, NF-kB transcription factor family members do not translocate to the nucleus to regulate gene transcription, and normal development of the organism, including the immune system, does not occur. In addition to these developmental defects, the immune system does not function correctly if NEMO is defective. Individuals with specific mutations that result in NEMO immunodeficiency and inflammatory disease are investigated using molecular, cellular and biochemical assays/techniques. Because NEMO syndrome is associated with defects in development of ectodermal structures in addition to secondary lymphoid structures in association with certain mutations, we seek to understand the function of NEMO in both of these roles in addition to its role in hematopoietically derived cells. In addition to clinical and laboratory evaluation of human patients, these studies are conducted using patient-derived peripheral blood cells, induced pluripotent stem cells and their derivatives, such as mesenchymal stem cells. A reconstitution system utilizing NEMO-deficient Jurkat T cells and THP-1 monocytes is also employed in order to study protein interactions biochemically. NEMO is required for canonical IKK activation, and in addition to IKK, recruits other signaling proteins such as TBK1 to activated receptors in order to induce the type I IFN response. The regulation of NF-kB activation by NEMO is complex: NEMO splice isoforms exist, and NEMO is post-translationally modified by serine phosphorylation, non-degradative ubiquitination, and sumoylation. We evaluate patients at the NIH Clinical Center to be able to comprehensively characterize the spectrum of phenotypes, and are working to develop protocols to offer advanced treatment based on our clinical and laboratory findings. In a similar fashion, we are studying individuals with NEMO-Like Syndrome who have suspected monogenic diseases which phenocopy NEMO syndrome in some aspects. We hypothesize that these individuals will have defects in signaling proteins or other regulatory proteins that interact in the NF-kB signaling pathway. Our general approach has been to discover candidate gene genes that may underly disease by sequencing exomes or genomes of affected individuals and their families. Candidate genes identified are evaluated using assays that interrogate different checkpoints along the NF-kB activation pathway. Insights into the role of proteins gained from study of patients with rare diseases will be applied to evaluation of the regulation of the immune system and immune response as it functions in the population, and in common inflammatory diseases such as arthritis and vasculitis.